Effective control of lung cancer continues to remain a clinical challenge resulting in poor five-year survival rate. Currently available therapies while showing promise have had limitations. Therefore, continued efforts for developing new therapeutic agents and systemic treatment modalities are warranted for treating lung cancer. This application addresses focuses on testing an improved interleukin (IL)-24 gene-based non-viral therapeutic for cancer. The PI?s laboratory has identified by modifying a phosphorylation (p) site in the wild-type IL-24 tumor suppressor/cytokine gene, the antitumor activity is improved and enhanced. Preliminary studies demonstrated replacement of a specific phosphorylation site in the wild-type IL-24 cDNA produced IL-24 protein (herein referred to as IL-24mt) that exhibited increased intracellular protein stability, secretion, and enhanced antitumor activity against lung cancer cells when compared to wild-type IL-24 (IL-24wt). Furthermore, inhibition of Gli1, and PD-L1 by IL-24wt both of which are known to support tumor growth, drug resistance, and metastasis was observed. Combinatorial approach with Gli1 inhibitor produced greater inhibitory activity on tumor cell growth, migration and invasion compared to individual treatments in vitro. Next, for applying IL-24-based therapeutic in vivo we adopted a non-viral delivery approach and used a cationic lipid-based nanoparticle (NP) system. The NP was decorated with a tumor-targeted ligand such as transferrin (Tf) for selective delivery of IL-24mt to tumor depots. Bio-distribution studies demonstrated TfNP preferentially accumulated in subcutaneous tumor and lung metastasis. Further, systemic administration of IL-24 contained in TfNP (IL-24-TfNP) in mice demonstrated a marked delay in lung tumor growth. To our knowledge, apart from our own observation reported herein, there are no prior reports demonstrating IL-24mt exhibited improved and enhanced anticancer activity over IL-24wt and it?s testing as a cancer therapeutic for lung cancer. On the basis of our new findings, we hypothesize that IL-24mt will demonstrate superior anticancer efficacy over IL-24wt both in vitro and in vivo that will be further enhanced when combined with inhibitors against Gli1, or PD-L1. To test our hypothesis we have identified three specific aims: Aim 1. Conduct biologic and molecular studies of IL-24mt and demonstrate its superior antitumor activity over IL-24wt in vitro. Aim 2. Deliver IL-24mt contained in TfNP and demonstrate the improved therapeutic efficacy in murine and human tumor xenograft tumor models. Aim 3. Conduct IL-24mt combinatorial studies with inhibitors against Gli1, and PDL1 in in vitro and in vivo tumor models. Demonstrating improved efficacy for IL-24mt over IL-24wt will lead to advanced studies aiding in clinical translation. Our findings will also have application against broad-spectrum of human cancers.